Drug – bio-affecting and body treating compositions – Enzyme or coenzyme containing – Hydrolases
Patent
1988-04-18
1991-09-10
Stone, Jacqueline
Drug, bio-affecting and body treating compositions
Enzyme or coenzyme containing
Hydrolases
435 712, 435223, 435814, 435896, 426 2, 426801, 426807, A61K 3754, C12N 952, A23K 1165
Patent
active
050472402
DESCRIPTION:
BRIEF SUMMARY
In the French Patent No. 2,034,559, the applicant described a process for producing proteolytic complexes starting from Streptomyces fradiae, and the possible use of such complexes especially in zootechny.
Experiments showed that the results obtained when the process of this patent is performed are generally positive but irregular or insufficient, so that the uses of such complexes remained very restricted.
The present application provides a process which enables one to obtain a new complex particularly having a more precisely stated composition and to define the conditions which allow this new complex to give with regularity some important improvements in zootechny.
The present application also provides the new proteolytic complex obtained according to this process.
In the English Patent No. 1,133,579, SHIONOGI mentioned that Streptomyces fradiae produces at least 5 proteases, named: Ia, Ib, II, III, IV and 2 peptidases. The applicant also obtained, starting from Streptomyces fradiae, some enzymes, which, having practically the same general properties, are of the same type as those described in the above SHIONOGI's Patent. So, in the present application, for the clarity of the language, the enzymes are designated with the same Roman numerals as those used in the said SHIONOGI's Patent.
The applicant observed that complexes which are exclusively composed with proteases of type II, III, IV give a moderate reduction of the intestinal mucus viscosity prescribed in its initial patent and generally give positive results in zootechny, but irregular or insufficient; Among all these complexes, only the particular complex, characterized by a greater abundance of protease of type II with regard to proteases of type III or IV, may regularly give an important improvement of zootechnic performance.
The applicant also observed that global complexes composed of the 5 types of proteases and the 2 types of peptidases, causes an excessive reduction of the intestinal mucus viscosity and are aggressive for the mucus of the membrane; they are then unusable in zootechny.
It is this reason why the applicant tried to obtain the particular complex exclusively composed of proteases of type II, III, IV with preponderance of protease of type II which is the object of claims 5 to 7 of the present application.
Process of Production
The complex, according to the invention, is obtained under the following conditions:
Fermentation
There is used a Streptomyces fradiae strain, for example the typical WASKMAN 3535 strain, filed at American Type Culture Collection (in abbreviated form: A T C C) under the number : 10,745. This strain may be periodically regenerated by the classic technique of dissociation on gelosed medium to eliminate possible spontaneous mutants having a low proteolytic activity. This strain may also be improved by the classic technique of induced mutation in order to obtain an increase of the proteolytic activity.
To prepare precultures of this strain, first in stirred flasks, then in an intermediate fermentor which is then inoculated in the fermentor of industrial production. The production medium may be composed as follows: soybean meal 15 g/l, glucose 30 g/l, bipotassium phosphate 1 g/l, and calcium carbonate 10 g/l. After sterilization, the pH is about 7.0. The temperature of fermentation is maintained at 28.degree. C. and the sterile air at 0.3 volume per volume of the medium per minute.
The pH of the medium, initially near 7.0, starts to decrease and reaches 6.5 after about 10 hours of fermentation; it increases then quickly and reaches 7.5 after about 20 hours of fermentation, then it continues to increase more slowly to finally reach 8.2.
There has been observed that it was very important to maintain the pH between 7.5 and 7.0 with an automatic addition of an acidic solution, for example, a 3N hydrochloric acid solution, which allows:
to avoid the fragmentation of mycelium and the release, in the medium, of some secondary metabolites, proteases of type Ia, Ib and peptidases. These secondary metabolites and the
REFERENCES:
patent: 3683069 (1972-08-01), Hooreman
patent: 3823072 (1974-07-01), Hooreman
patent: 4038419 (1977-07-01), Ferns
Shishkova et al., Chem. Abstracts, vol. 94: 204352s (1981).
Rassulin et al., Chem. Abstracts, vol. 94:204353t (1981).
Williamson et al., Biochimica et Biophysica Acta, 543 (1978), 397-402.
Kokubu et al., Biotechnol. Bioeng., vol. 13(1981) pp. 29-39.
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